Project Summary ATP was first discovered to be released from the urinary bladder epithelium (urothelium) in response to bladder distension in 1997. In the years since, many studies have determined additional stimuli that result in ATP release, however relatively few studies have examined the mechanism(s) of this release. As it is commonly thought that urinary ATP plays a significant role in the sensory pathways that control micturition, a deeper understanding of urothelial ATP release is essential for understanding urinary bladder physiology/pathology. We have recently discovered two distinct mechanisms controlling ATP release from the urothelium. The first involved pannexin hemi-channels, a type of large-pore ion channel permeable to ATP. We believe that this mechanism plays a significant role in the physiological control of micturition, as pharmacological inhibition or genetic knockdown of pannexin channels causes a marked inhibition of reflex bladder activity in the rat. The second mechanism involves secretory lysosomes. We have evidence that this mechanism plays a role in the emergence of bladder inflammation, as lysosomal exocytosis can be stimulated by bacterial endotoxins. This has led us to hypothesize that the ATP released through different mechanisms may have separate physiological effects. The next step into fully understanding ATP's role in bladder physiology and pathology is to fully characterize the intracellular signaling pathways responsible for activating or inhibiting either release mechanism. To that end, we propose to use known activators of either pannexin mediated release (bladder distension, ?3 nicotinic receptor stimulation and P2Y6 purinergic receptor stimulation) or lysosomal mediated release (toll-like receptor stimulation) to examine common intracellular signaling pathways that may control release. For example, it is already known that pannexin mediated release is dependent on intracellular calcium signaling and activation of the RhoA/ROCK pathway. Our current research aims to determine if each stimulus (mechanical stretch, activation of an ion channel and activation of a metabotropic receptor) feeds into these known intracellular pathways. We will also examine how release of lysosomal calcium following NAADP signaling or alterations in lysosomal pH, pathways known to modulate lysosomal exocytosis in other tissues, alter lysosomal release of ATP from the urothelium. Finally, we will characterize how the two ATP release mechanisms interact, as we have previously demonstrated that inhibition of pannexin-mediated release potentiated lysosomal release, suggesting a crosstalk between release mechanisms. It is our hope that this project will lead to a more complete understanding of the mechanisms controlling purinergic signaling in the urinary bladder and lead to more effective treatments for bladder pathology.